We and others have reported the isolation of a clone encoding for the CCAAT/Enhancer Binding Protein (C/EBP)I3 (LAP, NF-IL6, IL6-DBP), a liver-enriched activator protein that confers liver-specific gene expression. This proposal will focus on the signal transduction pathways that modulate hepatocyte proliferation and survival through site-specific phosphorylations of C/EBP!3. We have found that transforming growth factor (TGF)-c_ and phorbol esters/protein kinaseC (PKC)-o_ mediate their effects on hepatocyte proliferation, through the activation of ribosomal S6-kinase (RSK)- 2 and phosphorylation of C/EBPI3 on its activation domain. We have established that C/EBP(3 affects the GI/S phase transition of the cell cycle. Moreover, we found that this phosphorylated C/EBP!3 plays a major role in cell survival by binding to procaspase 8 and preventing its self-cleavage and activation. Our recent discovery identifies a novel non- transcriptional role of C/EBPI3PThr z_7as the first mammalian protein acting as an inhibitor of caspases. As expected, we have found that the nonphosphorylatable C/EBP_ Ala217mutant prevents hepatocyte proliferation induced by the TGFc_/ERK/MAPK/RSK signaling cascade, while the phosphorylation mimic C/EBPI3Glu 2_zmutant blocks hepatocyte apoptosis initiated by death receptors or proteasome inhibitors (FAS/caspase 8 pathway).
The specific aims of this proposal are to assess: 1. The regulation of the cell cycle by C/EBPI3 in hepatocytes. 2. The regulation of hepatocyte proliferation and tumorigenesis in C/EBPI3 Ala2_7transgenic mice. 3. The role of C/EBP_ phosphorylation on hepatocyte survival. 217 4. The modulation of hepatocyte cell survival in C/EBPI3 Glu transgemc mice. 5. The effects of C/EBPI3Glu 217peptides on hepatocyte survival. 6. The role of C/EBPI3 phosphorylation on the development of hepatocellular carcinoma in patients. These studies are relevant to the hepatocyte proliferation and survival characteristic of many physiological and pathological conditions, including hepatocellular injury, liver regeneration and hepatocellular carcinoma.
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